The clinical trial component of this proposal consists of a phase I study of lentiviral vector transduced T-cells expressing anti-HIV siRNA (J. Zaia, P.I.). This will be the first trial of anti-HIV siRNA in humans and will be performed at City of Hope with collaboration from C. June at UPENN and M. Jensen at COH. The goals of the study are to determine the feasibility and safety of lentiviral transduction and expansion of T-cells from AIDS patients who have failed HAART. The preclinical and the clinical aspects of T-cell function will be characterized by C. June after transduction and expression of anti-HIV RNAs. He will provide important GMP manufacturing scale up support for the clinical trial in Project 5 in the areas of expansion technology and analysis of the function of T-cell after transduction and selection. In anticipation of a T cell selection system, the effect of IMPDH on T cell function and the safety of lentivirus integration will be explored. The specific aims are as follows: Aim 1: To determine the feasibility and safety of RNAi-lentivirus transduced T cell immunotherapy in the setting of AIDS: Study 1: The lentivirus vector, pHIV7-shII, has been selected for clinical phase I studies based on comparative anti-HIV effect in vitro (see PRELIMINARY RESULTS). This vector was developed in our current IPCP activity by J. Rossi and J.K. Yee, and it is a self-inactivating lentivirus vector encoding a short hair-pin RNAi targeting an exon in HIV-1 rev. The clinical grade T cell product will be characterized for immune function, TCR repertoire, and resistance to HIV before and after infusion into research patients. In this study, the T cell collection, transduction, and expansion will be performed at BRICOH in Core C, and the subjects will be treated in the GCRC. The biologic effect of this transduction on cells pre- and post-infusion will be evaluated. Study 2: To determine the safety and relative efficacy of a lentivirus encoding a multiplex RNAi developed in this IPCP. Using the first generation pHIV7-shlI lentivirus vector evaluated in aim 1 for comparison, a second clinical trial will evaluate the relative survival of T cells transduced with a multi-plexed RNAi vector developed in Projects 1-3. In this second clinical trial, subjects will receive combined T cells transduced with either the fast generation RNAi vector or with the improved vector and then observed for relative T cell survival in the presence of HIV. Aim 2: To characterize the function of T cells after in vitro expansion and selection: The immunologic function of T cells expanded after lentivirus RNAi transduction and after transduction with new vectors developed in Projects 1-3 will be studied. A particular focus of this work will be the effect of T cell selection and expansion, using either IMPDH2- or MGMT-based methods, on the immunologic function of these cells in vitro and on the anti-HIV effect. This will contribute to the safety phase of study #1 and to the pre-clinical phase of study #2.